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Full text of "Vitamin A and the interferon system"

VITAMIN A AND THE INTERFERON SYSTEM 



By 



JAMES EDWIN BLALOCK / 9 '/V- 



A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF 
THE UNIVERSITY OF FLORIDA 
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE 
DEGREE OF DOCTOR OF PHILOSOPHY 



UNIVERSITY OF FLORIDA 
1976 



ACKNOWLEDGEMENTS 

This dissertation is dedicated to my parents, Mr. Maury J. and 
Mrs. Elizabeth B. Blalock and to other members of ny family for their 
love and belief in me. 

1 extend my sincere appreciation to my teacher and friend, Dr. 
George E. Gifford, for his constant willingness to help and guide me 
through my graduate years. I especially thank him for always encouraging 
me to be scientifically inquisitive and for firmly establishing, by 
example, that there is a right way to do science. I am especially 
grateful to Dr. Donna H. Duckworth for her friendship and genuine 
interest in my well-being. I also thank the other members of my 
advisory committee, Dr. J.W. Shands, Jr., Dr. L.W. Clem, Dr. Kenneth Ley, 
Dr. P. A. Klein and Dr. P. A. Small, Jr. for their suggestions and 
encouragement during my studies. Special thanks also go to the other 
members of the faculty, especially Dr. R.B. Crandall, and staff of the. 
Department of Immunology and Medical Microbiology, who have helped me 
in many ways. 

I am grateful to my former and to my present fellow graduate students 
for their suggestions and support throughout this investigation. In 
particular, I thank Rick Weber and Dave Dion for the many scientifically 
stimulating conversations. 

Finally, I would like to express my gratitude to Mr. Mike Duke and 
Mr. Joe Brown for their excellent technical assistance and for making 
Dr. Gic ford's laboratory such a fun place to work. 



1 L 



TABLE OF CONTENTS 

ACKNOWLEDGEMENTS ,,,....,.. ii 

LIST OF TABLES c iv 

LIST OF FIGURES , « . • v 

KEY TO ABBREVIATIONS vi 

ABSTRACT vii 

INTRODUCTION = 1 

MATERIALS AND METHODS 5 

Materials 5 

Methods <> 7 

RESULTS 11 

Vitamin A and Interferon Action 11 

Vitarain A and Interferon Production 29 

Structural Requirements of Vitamin A for 
Suppression of Interferon Production and 

Inhibition of its Action. ... c .. . 56 

DISCUSSION 59 

LITERATURE CITED 68 

BIOGRAPHICAL SKETCH „ 73 



xxx 



TABLES 



Table 

1. Effect of Bovine Serum Albumin on the Simultaneous 
Addition of Retinoic Acid and Interferon to Cells 17 

2. Effect of Retinoic Acid on Interferon and 

Cellular RNA Synthesis 30 

3. Effect of Calf Serum on Suppression of Cellular 

RNA Synthesis by Retinoic Acid 32 

4. Effect of Retinoic Acid on L-929 Cell Cultures 34 

5. Effect of Retinoic Acid on L-929 Cell Proliferation 35 

6. Effect of Retinoic Acid Concentration on Interferon 
Production by NDV 36 

7. Effect of Retinoic Acid on the Intracellular Level 

of Interferon 38 

8. Effect of Retinoic Acid on NDV, SFV and Poly I:C 
Induction of Interferon 40 

9. Effect of Time of Treatment with Retinoic Acid 

(20 ug/ml) after NDV Adsorption 42 

10. Effect of Cyclohexitnide on Interferon Production 50 

11. Effect of Cycloheximide on Suppression of 

Interferon Production by Retinoic Acid 52 

12. Structural Requirements of Vitamin A for 
Suppression of Interferon Production and 

Inhibition of Its Action 57 



IV 



FIGURES 
Figure 

1. Effect of simultaneous addition of retinoic. acid and 
interferon on the assay of interferon " 

2. Effect of calf serum on the simultaneous addition of 
retinoic acid and interferon to cells -*-*> 

3. Effect of treatment of interferon with retinoic acid 
prior to assay for activity of interferon ^0 

4. Effect of calf serum on the treatment of interferon 
with retinoic acid priot to assay for interferon 
activity " 



5. Effect of temperature on treatment of interferon with 

or 

retinoic acid J 

6. Effect of time at 37°C on the loss of interferon 

TO 

activity in the presence of retinoic acid 

7. Effect of time of addition of retinoic acid after NDV 
adsorption ^ 

8. Effect of time of addition of retinoic acid after 
addition of poly I : C ^ 6 



9. Kinetics of interferon production by control and 
retinoic acid treated cells 

10. Kinetics of interferon action in the presence of 



retinoic acid 



v 



KEY TQ ABBREVIATIONS 

BSS.., o Balanced salt solution 

CS o . . . . <■ c . . , Calf serum 

DEAE-dextran ..„o. Diethylaminoethyl dextran 

DMSO o . . - o . o Dimethyl sulfoxide 

DNA .c.... Deoxyribonucleic acid 

HA Hemagglutination 

MEM , Minimal essential medium 

m-RNA Messenger ribonucleic acid 

NDV. .„ Newcastle disease virus 

PBS „ „ Phosphate buffered saline 

PDD50 50% plaque depressing dose 

PFU . Plaque forming unit 

Poly I:C Polyriboinosinaterpolyribocytidilate 

RNA. Ribonucleic acid 

SFV Semliki Forest virus 

TCA. «... Trichloroacetic acid 

VSV Vesicular stomatitis virus 

V/V Volume per volume 



V L 



Abstract of Dissertation Presented to the Graduate Council 
of the University of Florida in Partial Fulfillment of the Requirements 
for the Degree of Doctor of Philosophy 



VITAMIN A AND THE INTERFERON SYSTEM 
By 
James Edwin Blalock 
June, 1976 

Chairman: Dr. George E. Gifford 

Major Department: Immunology and Medical Microbiology 

Vitamin A was shown to suppress both interferon action and production. 
The mechanisms of the two inhibitory effects of vitamin A appeared to be 
different and to occur by different moieties of the vitamin A molecule. 
The inhibition of interferon action seemed to result from an interaction 
of vitamin A with the interferon molecule. The loss of interferon 
activity was characterized by a dependence on time and temperature and 
was prevented by calf serum. The suppression of interferon production 
v/as due to an effect of vitamin A on the cell. The kinetics of the suppres- 
sion and results from experiments with metabolic inhibitors are consistent 
with transcription of the interferon gene being blocked by a vitamin A— 
induced protein. Our data, therefore, point to a site of action of 
vitamin A at the genetic level and our system provides a potential 
model for the study of control of gene expression by vitamin A. 



VJ.1 



INTRODUCTION 
Vitamin A is an essential nutrient for vertebrates. Many of the 
known physiological actions of this vitamin have been elucidated by 
studying the effects of vitamin A deficiency. In its absence, animals 
suffer from blindness, -*■ retarded growth, 2 impaired reproductive 
capacity 3 an d ultimately death. At the tissue and cellular level 
some of the most profound alterations associated with hypovitaminosis A 
involve epithelial structures. About 50 years ago Wolbach and Howe 
observed that vitamin A deficiency caused a replacement of mucus-secreting 
epithelia by stratified keratinizing epithelia. In this classic study, 
vitamin A was postulated to induce and control epithelial differentiation. 
This hypothesis was strengthened when in 1957 Fell substantiated the 
findings of Wolbach and Howe by demonstrating that excess vitamin A 
in vitro caused keratinizing tissues to become mucus-secreting. More 
recently, vitamin A has been observed to have both a prophylactic and 

therapeutic effect on several types of experimental tumors. For 

6 7 

instance, Saffiotti _et al. and Bollag showed a suppressive effect 

of vitamin A on the induction and development of tumors in response 

o 

to chemical carcinogens. In another system, Felix et_ _al. demonstrated 
an anti-tumor action of vitamin A in mice inoculated with murine melanoma 
cells. Although other explanations are possible, the anti-tumor quality 
of vitamin A might also be interpreted as a reflection of the ability 
of vitamin A to induce and control differentiation. Our laboratory 
has, in fact, observed an apparent restoration of the control of pro- 
liferation of a transformed cell line in vitro by this vitamin. 



In spite of the impressive amount of knowledge on the physiological 
effects of vitamin A, its mechanism (s) of action remains obscure, 
with the exception of its role in vision. At the. biochemical level 
a number of systems have been studied in the search for the mechanism(s) 

of vitamin A action. For instance, there is no doubt that vitamin A 

1 ? 
labilizes membranes. A hallmark of this labilization is the extra- 
cellular release of lysosomal enzymes by cells treated with vitamin A. 
In vitro , fetal cartilage or bone underwent resorption in the presence 
of vitamin A. 13 This resorption was surmised to be caused by a 
lysosomal acid protease, whose extracellular release was shown to be 
increased by vitamin A. - 1 Isolated lysosomes from liver, also, 
rapidly released proteases upon addition of vitamin A. 15 similar effects 
occur in vivo. Hypervitaminosis A in rabbits resulted in dissolution 
of cartilage. This condition also caused release of the liver 
lysosomal enzyme, g-glucuronidase, into serum. 1 These effects, while 
indicating a role in membrane function, only seem applicable in a 
hypervitaminotic state. 

Vitamin A has also been postulated to serve a coenzyme function and 
that enzymatic conversion of essential substances is suppressed in 
vitamin A deficiency. 18 Hence, vitamin A deficiency resulted in de- 
creased activities of gulonolactone oxidase, codeine demethylase, 
A 5 - 3 B-hydroxysteroid dehydrogenase, 21 sulfate transferase, ' " and 
ATP-sulfurylase. 23 However, in the ATP-sulfurylase system, 2i which 
was the most rigorously studied, it seems that the decreased activity 
was the result of less of the enzyme rather than the absence of a co- 
enzyme function of vitamin A. Whether the diminished amount of enzyme 
was due to increased catabolism or decreased synthesis is not known. 



20 
Interestingly, the decreased activity of codeine demethylase 

observed in vitamin A deficiency was reversed by the addition of vitamin A 
and actinomycin D inhibited the reversal. Since actinomycin D blocks 
DNA dependent RNA synthesis, this data suggests de novo synthesis of 
the enzyme in response to the vitamin and could be interpreted as genetic 
transcription being one possible site of vitamin A action. Consistent 
with this idea, is the observation that vitamin A stimulated RNA syn- 
thesis of intestinal mucosa 2 ^ and liver 25 from vitamin A deficient rats. 
Incorporation of radio-labeled uridine was especially increased in the 
nuclear fraction and the increase occurred within 5 minutes. J De- 
creased protein synthesis was also associated with vitamin A deficiency. 
This, however, could be explained as a secondary effect of blocked 
transcription. Perhaps, a transcriptional block by vitamin A deficiency 
could account for all of the depressed enzyme activities. There have 

9 7 9& 
also been reports of vitamin A induced glycoprotein synthesis. Cl * The 

specific biological functions of these glycoproteins are unknown. 

The obvious question which arises from the above mentioned apparently 
diverse observations is whether they are mediated by a number of modes 
of action of vitamin A or by a single action on a cardinal function, 
such as transcription. A key role for vitamin A in control of gene 
expression seems quite feasible and is certainly attractive. It would, 
therefore, be advantageous to study the effect of vitamin A on an 
inducible cell system with a known biological function. 

The interferon system is a normal defense mechanism of an animal 
or animal cell to virus infection. Virus infection of cells leads to 
the induction, synthesis, and release of a new cellular protein, inter- 
feron. 2 9 Interferon molecules then interact with neighboring, uninfected 



Of) Ol 

cells and induce another cellular protein, the antiviral protein. JW ' J - L 
It is the antiviral protein, in turn, that causes these cells to become 
resistant to subsequent virus infection. One can reasonably conclude, 
a priori, that control at the transcriptional level exists for interferon 
since it is synthesized de nov o. Additionally, a cell culture after one 
exposure to an inducer, will not produce the usual amount of interferon 
upon reexposure to an inducer during the next several days . This hypo- 
reactivity or feedback inhibition of interferon synthesis may also re- 
present transcriptional control. Employing the same reasoning as for 
interferon synthesis, the synthesis of the antiviral protein seems to be 
controlled at the transcriptional level since it is synthesized de novo . 
The interferon system can, therefore, be divided into interferon pro- 
duction and interferon action which share the common feature of induci- 
bility. This common feature provides a unique means of study of the 
effect of vitamin A on the induction of cellular functions. 

Initially, we observed an inhibitory effect of vitamin A on human 

on 

interferon action. J " In the present study, we have extended this 
observation to include the inhibitory effect of vitamin A on mouse 
interferon action. We have further shown a suppressive effect of this 
vitamin on mouse interferon production. The mechanisms of these two 
effects on the mouse interferon system were shown to be different and 
appeared to involve different moieties of the vitamin A molecule. 



MATERIALS AND METHODS 

Materials 

Virus Strai ns 

Vesicular Stomatitis Virus (VSV) . A large plaque variant of the 

v 

Indiana strain was obtained from Dr. Jan Vilcek, New York University. 

Newcastle Disease Virus (NDV) . A lentogenic strain was obtained 
from Dr. R.P. Hanson, University of Wisconsin. 

Se mliki Forest Virus (SFV) . Kumba strain was obtained from Dr. 
J. Porterfield, National Institute for Medical Research, London, England. 

Cell Cultures 

Mouse L cell cultures . Strain 929 

Media 

L cell growth media . Eagle's Minimal Essential Medium (MEM), 

obtained from International Scientific Industries (Cary, Illinois), 
was supplemented with 10% calf serum (CS) . This medium contained 
125 yg streptomycin and 250 units of penicillin/ml. 

M ethyl cellulose overlay . The overlay medium for plaque assay 
consisted of MEM containing 1% methyl cellulose (1500 centipoise, Fisher 
Scientific Company), 5% CS, 25 mM N-2-hydroxyethylpiperazine-N-2 -ethane- 
sulfonic acid, 125 ug/ml streptomycin and 250 units/ml of penicillin. 

Balanced salt solution . Gey's balanced salt solution (BSS) was 
employed. 

Ph osphate buffered saline . Gey's BSS without calcium and magnesium 
salts was employed as phosphate buffered saline (PBS). 



Reag ent s 

Polyribonucleotides . The homopolymer pair polyriboinosinate: 
polyribocytidilate (poly I:C) was purchased from Sigma Chemical 
Company, St. Louis, Missouri. A stock solution of the polymer at a 
concentration of 1 mg/ml was prepared in MEM and stored at -20°C. 

Radioisotope . Uridine-5- H was obtained from Schwarz/Mann, 
Orangeburg, New York. The specific activity was 20 Ci/mM. 

Scintillation fluid . Radioactive samples were diluted in scintil- 
lation fluid containing 8.25 g of 2,5-dipheniyloxazole (Fisher 
Scientific Company), 0.25 g of l,4-bis-2-(4-methyl-5-phenylo- 
xazolyl) -benzene (Packard), 1,000 ml of toluene (J.T. Baker 
Chemical Company) and 500 ml of Triton X-100 (Packard). 

Actinomycin D . A stock solution of 100 ug/ml of actinomycin D 
(Grand Island Biological Company) was prepared in MEM and stored 
at -20°C. 

Cycloh eximide . Cycloheximide (Grand Island Biological Company) was 
dissolved at 1 mg/ml in MEM and stored at -20 °C. 

Diethylamiiioethyl (DEAE) dextran . DEAE-dextran (Pharmacia Fine 
Chemicals, Uppsala, Sweden) was dissolved at 10 mg/ml in MEM and stored 
at -20°C. 

Vitamin A and related compounds . All trans forms of retinoic acid 
(vitamin A acid), retinol (vitamin A alcohol), retinal (vitamin A 
aldehyde), and retinyl acetate (acetate ester of vitamin A) were purchased 
from Sigma Chemical Company. Vitamin K^ was obtained from Schwarz/Mann. 
Trans £-carotene and citronellol were obtained from Aldrich Chemical 
Company, Milwaukee, Wisconsin. Stock solutions were prepared by making 
each compound 6.7 X 10~ 3 M in dimethyl sulfoxide (DMSO, Fisher Scientific 



Company) and stored at -20°C. For experimental purposes, the compounds 
were diluted to 6.7 X lO - ^ M (equivalent to 20 yg/ml of retinoic 
acid) in culture medium. 

Methods 

Cell Cultures 

Mouse L cells (Strain 929) . Cell cultures were maintained and 
propagated in 32 oz prescription bottles in MEM with 10% CS. These 
cells were passaged weekly. The growth medium was decanted, the cell 
sheet was washed with PBS and treated with 0.1% trypsin and 0.04% versena 
in PBS for 30 seconds. When the cells had detached from the glass 
surface they were diluted in fresh growth medium and dispensed into 32 oz 

bottles for further propagation or into 2 oz glass bottles for production 

3 

and assay of interferon, assay of viruses and H-uridine incorporation. 

Cell counts . Cell monolayers were treated with 1 ml of 0.1% trypsin 
in phosphate buffered saline (PBS) containing 0.04% versene. After 
30 seconds, the trypsin solution was removed, and 3-4 minutes later, when 
cells detached from the glass surface, 4 ml of MEM with 5% CS was added. 
After addition of 1 ml of 0.5% trypan blue in buffered saline, to determine 
cell viability, an aliquot of the cell suspension was counted in a 
haemocytometer. Cells unable to exclude trypan blue dye were counted as 
dead. 

Growth and Assay of Viruses 

Preparatio n. NDV and VSV were grown in the allantoic cavity of 
10-day-old chick embryos by inoculation of 0.2 ml of a virus suspension. 
After 48 hours incubation at 37°C, the eggs were chilled and the allantoic 



fluid collected. The allantoic fluid was centrifuged at low speed 
(1,000 rpm) to sediment red blood cells and tissue fragments. The super- 
natant fluids were dispensed in sealed glass ampules and stored at 
-70°C. SFV was propagated in the brains of newborn mice. The newborn 
outbred mice were intracerebrally inoculated. The infected brains were 
harvested 48 to 72 hours after infection and a 10% suspension of brains 
in medium was made with a Potter-Elvehjem homogenizer. The suspension 
was centrifuged to remove coarse material. The supernate was dispensed 
in sealed glass ampules and stored at - 70°C. 

Assay . VSV and SFV were assayed on monolayers of L-929 cells by 
plaque assay. Medium was aspirated from monolayer cultures and cells 
were infected with 0.2 ml of a suitable dilution of virus. After 1 hour 
incubation at room temperature to permit virus adsorption, each monolayer 
was overlaid with 5 ml of methyl cellulose overlay. During the virus 
adsorption period cultures were rocked every 15 minutes to evenly distribute 
the virus suspension. The cultures were incubated for 48 hours at 37°C 
and monolayers were then stained with crystal violet. Assays were 
performed in triplicate or quadruplicate. Plaques were enumerated after 
X6.5 magnification of the monolayers by use of a photographic enlarger. 
By this assay the pools of VSV and SFV contained 3.0 X 10 9 and 2.1 X 10 8 
plaque forming units (PFU)/ml respectively. NDV was assayed by 
hemagglutination with chicken red blood cells. Following an initial 
1 to 10 dilution, two-fold serial dilutions of the virus suspension 
were made in hemagglutination buffer (Difco Laboratories, Detroit, Michi- 
gan). To 0.5 ml of diluted virus was added 0.5 ml of a 0.5% suspension 
of red blood cells. One hemagglutination (HA) unit was defined as the 



highest dilution of virus which gave, total hemagglutination. The pool 
of NDV contained 320 HA units/ml. 

Produ ction and Assay of Interferon 

Production . Interferon was prepared by inoculating monolayer cultures 
of L -929 cells with NDV or with SFV. Triplicate cultures were used for 
all determinations. After adsorption at room temperature for one hour, 
residual virus was removed, cultures were washed and fresh medium added. 
Culture fluids, unless specified, were harvested at 24 hours after 
infection, the triplicate samples pooled, and clarified by low speed 
centrifugation. Culture fluids were then dialyzed against pH 2 buffer 
for five days at 9°C and then against Gey's balanced salt solution (BSS) 
to restore pH to neutrality. 

Interferon was also induced by treatment of confluent monolayers of 
L-929 cells for various times with medium containing 10 ug/ml of 
poly I:C and 100 ug/ml of DEAE dextran. The medium was then removed, 
cells washed and refed fresh medium. Culture fluids were harvested 24 
hours after poly I:C treatment and assayed for interferon. 

Assay o A plaque reduction assay using VSV and L-929 cells was 
employed to determine the interferon content of an interferon preparation. 
Confluent monolayers in 2 oz glass bottles were treated overnight at 
37°C with twofold serial dilutions of interferon preparation. Dilutions 
of interferon were made in MEM supplemented with 5% CS. Supernatant 
fluids were then aspirated and cells infected with 0.2 ml of a dilution 
of VSV containing about 300 PFU. The remainder of the assay is 
identical with that used for titration of VSV. Assays were performed 
in triplicate or quadruplicate. The 50% plaque depressing dose (PDD50) 



10 



was defined as the amount of an interferon preparation, in ul, 
that inhibited 50% of the plaques from developing as compared to the 
controls. The PDD50 was calculated according to the method of Linden- 
mann and Gifford. 

-%-Uridine Incorporation 

Twenty yCi of J H-uridine in 0.1 ml of MEM were added for 1 hour 
to monolayer cultures in 2 oz glass bottles containing 2 ml of medium. 
Incorporation was stopped by decanting the supernates and placing 
the cultures on ice. Unincorporated label was removed by 5 successive 
washes of the cell sheets with 5 ml of cold 5% trichloroacetic acid 
(TCA) . Cultures were drained by inverting on absorbent towels and then 
2 ml of 5% TCA were added to each culture. Incorporated H-uridine was 
hydrolyzed by heating the cultures at 80°C for 1 hour. Aliquots 
(0.2 ml) of hot TCA extracts were placed in 10 ml of scintillation 
fluid and counted in a Beckman LS-230 liquid scintillation counter. 



RESULTS 
Vitamin A and Interferon Action 

Effect of Simultaneous Addition of Retinoic Acid and Interferon on the 

Assay of Interferon . 

Separate interferon assays were simultaneously performed in the 
presence or absence of different concentrations of retinoic acid 
(vitamin A acid) . Dilutions of interferon and retinoic acid in MEM 
with 3% calf serum (CS) were mixed and added to L cell monolayers 
which were then incubated overnight at 37°C. Residual interferon and 
retinoic acid were removed and cultures challenged with approximately 
300 PFU of VSV. Overlay medium was added and cultures incubated 
at 37 C C for 48 hours. Plaques in experimental and control cultures 
were counted and the percent inhibition of plaque formation was plotted 
against the logarithm of the dose of interferon in ul. A series of 
approximately parallel dose response lines were obtained. These 
were used to estimate the PDD50 units of interferon measured with each 
concentration of retinoic acid. The percentage of control PDD^q 
units obtained in the presence of various concentrations of retinoic 
acid were calculated for two separate experiments and are shown in 
Figure 1. Increasing concentrations of retinoic acid resulted in 
decreasing activity of interferon. A marked reduction (93%) in 
measurable, interferon xras obtained when 20 yg/ml of retinoic acid 
was employed (from 6.7 PDD5Q units in the control to 0.5 units). This 
concentration of retinoic acid was not demonstrably toxic for the cells 
since the same number (and similar size) of VSV plaques were found with 
all concentrations of retinoic acid (and controls) in the absence of 



11 



Figure 1. Effect of simultaneous addition of retinoic acid and 
interferon on the assay of interferon. Separate 
interferon assays were simultaueously performed in 
the presence or absence of different concentrations 
of retinoic acid in MEM with 3% CS. The percentage 
of control PDD50 units (6-7 units) obtained in the 
presence of various concentrations of retinoic acid 
were calculated for two separate experiments (X or ($) 



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interferon. Greater concentrations of retinoic acid could not be used 
since they were toxic for the L cells. 

Effe ct of Calf Serum or Bovine Serum Albumin on the Simultaneous 
Addition of Retinoic Acid and Interferon to Cells . 
Preliminary experiments indicated that the effect of retinoic acid 
on the interferon assay was markedly influenced by the concentration 
of calf serum employed. Various concentrations of calf serum (%, v/v) 
were employed with 20 yg/ml of retinoic acid or 1% DMSO and a dose of 
interferon which would normally result in approximately 80% inhibition 
of VSV plaques. Controls without interferon received an equivalent 
amount of retinoic acid or DMSO. These mixtures were then assayed 
for determination of resultant interferon activity. These results 
(Figure 2) indicate that concentrations of calf serum from 2.5 to 
20% had only a slight effect on the interferon assay as previously 
reported by Vilcek & Lowy. 34 Hoxrever, increasing concentrations of 
calf serum decreased the inhibitory effect of retinoic acid on inter- 
feron action. 

In a similar experiment, bovine serum albumin was substituted for 
calf serum. Table 1 shows that increasing concentrations of bovine 
serum albumin also decreased the inhibitory effect of retinoic acid 
on interferon action. 

E ffect of Trea tme nt of Interferon with Retinoic Acid Prior to Assay 

for Activity of Interferon . 

A loss of measurable interferon activity, when the interferon is 
assayed in the presence of retinoic acid, could be due to either a 
combination of interferon with retinoic acid resulting in an inactive 



Figure 2. Effect of calf serum on the simultaneous addition of 
retinoic acid and interferon to cells. Vertical bars 
indicate the standard deviation of quadruplicate 

determinations; & @, interferon; O- ~ ~0> interferon 

plus retinoic acid (20 yg/ml) . 



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% Inhibition of 
Control Interferon Activity 


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% Inhibition of 

VSV Plaques 

(Standard Deviations) 


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product or to some intracellular event, influenced by retinoic acid, 
which prevented the expression of interferon activity. The previous 
data showing that increasing concentrations of calf serum or bovine 
serum albumin prevented the effect of retinoic acid on interferon 
activity suggested that the event was extracellular. To test this 
possibility, interferon and retinoic acid were mixed, incubated, and 
then diluted beyond the effective range of retinoic acid but with 
sufficient interferon remaining to significantly inhibit virus repli- 
cation. Thus, a 1:10 dilution of stock interferon in MEM was mixed 
with various concentrations of retinoic acid. Control interferon 
received an equivalent amount of DMSO. Since the stock interferon 
was made in 10% calf serum, the resultant concentration of calf serum 
employed was 1%. Following overnight incubation (22 hours) at 37°C, 
interferon activity was assayed. Prior to assay, the interferon samples 
were sufficiently diluted in MEM with 5% CS so that the residual re- 
tinoic acid was diluted beyond the concentration needed to interfere 
with the assay (i.e. 0.4 to 0.003 ug/ml) . The control activity 
represented 400 PDD50 units of interferon. Figure 3 shows that retinoic 
acid treatment of interferon resulted in a loss of interferon activity 
under these conditions. This data indicates that interferon and re- 
tinoic acid must interact in some fashion to inhibit interferon 
activity and that this interaction was not rapidly reversible. 

Effec t, of Calf Serum on the Treatment of Interferon With Retinoic Acid 

Prior to Assay . 

Since the concentration of calf serum markedly influenced the 
effect of retinoic acid on the interferon assay when assayed immediately 



Figure 3. Effect of treatment of interferon with retinoic acid 
prior to assay for activity of interferon. The 
control activity represents 400 PDDcq units of 
interferon. 



20 



100 



!E 
< 

o 

&_ 
o 

•a— 



c: 
O 
O 




10 20 30 40 

Concentration of Retinoic Acid (ajg/ml) 



21 



(see Figure 2) , the effect of calf serum was then determined on the 
interaction of retinoic acid and interferon when incubated together in 
the absence of cells. 

A dilution of the stock interferon was made in MEM with 1, 5 or 
10% calf serum and with or without 20 yg/nJ of retinoic acid. Following 
overnight incubation (22 hours) at 37°C, interferon activity was 
assayed after dilution (residual concentration of retinoic acid following 
dilution was less than 0.2 pg/ml). Figure 4 shows that calf serum 
again prevented an apparent interaction of interferon and retinoic acid 
under these conditions. In another experiment the calf serum concen- 
tration was reduced to 0.125%, and interferon activity was thereby 
reduced to 2.5% (10 units) of the control (400 PDD5Q units/ml) when 
40 yg/ml of retinoic acid was employed. 

Effect of Temperature on Treatment of Interferon With Retinoic Acid . 

Since it appears that interferon and retinoic acid interact to 
result in an inactive product, the effect of temperature on this 
interaction was determined. 

Retinoic acid was added to interferon in MEM (400 units/ml in 1% CaS) 
at a concentration of 20 jig/ral. One ml portions of the interferon and 
retinoic acid mixture, as well as control interferon prepared without 
retinoic acid, were placed at 5, 25 and 37°C for 24 hours. The 
mixtures were then diluted 1:100 in MEM with 5% CS (reducing the retinoic 
acid concentration to 0.2 ug/ml and assayed for residual interferon 
activity. Figure 5 shows only a slight loss of interferon activity 
in the control at 37°c, while there is a narked loss of inter- 
feron activity at this temperature in the presence of retinoic acid. 
No loss of interferon activity resulted when the mixture of interferon 



Figure 4. Effect of calf serum on the treatment of interferon 
with retiaoic acid prior to assay for interferon 

activity; © ^ interferon; O Q "retinoic 

acid-treated interferon. 



23 



400-j 



Q 



300 J 



o 

o 

o 

I/) 
E 

o 

ID 
Q 
Q 



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CO 



200- 



100- 







t 



I 



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7 



/ 



/ 



/ 



/ 



/ 



/ 



© • Interferon 

° ° Retinoic Acid Treated 

Interferon 







"T 

5 



10 



Concentration of Calf Serum (%) 



Figure 5. Effect of temperature on treatment of interferon with 
retinoic acid. Vertical bars indicate the standard 
deviation of quadruplicate determinations; © ^ inter- 
feron; O" _ "O retinoic acid-treated interferon. 



25 



in 
o 






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90- 



80- 



70 



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cr. 



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Interferon 



o o Retinoic Acid Treated Interferon 



T 



10 15 20 25 

Temperature (°C) for 24 hours 



30 



-T-G 
35 



26 



and retinoic acid were kept at 5°C for 24 hours. This data would 
indicate that the interaction of interferon and retinoic acid resulting 
in the loss of interferon activity was temperature dependent. 

Effect of Time at 37°C on the Loss of Interferon Activity in the 

Presence of Retinoic Acid . 

Retinoic acid (20 ug/ml) was added to 400 units /ml of interferon 
in MEM (1% CS) . The mixture was placed at 37°C and portions were 
removed at various times and placed at 5°C (a temperature at v/hich 
retinoic acid did not result in a loss of interferon activity) . when 
all samples were collected (23 hours), each sample was diluted 1:100 
in MEM with 5% CS (reducing the retinoic acid concentration to 0.2 ug/ml) 
and assayed for residual interferon activity. As shown in Figure 6, 
there is a progressive loss of interferon activity in the presence of 
retinoic acid with increasing periods of time at 37°C. 



Figure 6. Effect of time at 37°C on the loss of interferon 

activity in the presence of retinoic acid. Vertical 
bars indicate the standard deviation of quadruplicate 

determinations; ® O, interferon; ^ -@, retinoic 

acid-treated interferon. 



28 



lOOn 






cr 
a 



> 

CO 

> 



c 
o 



X) 
x; 

C 




50- 



40- 



30- 



20- 



is 



N 



• • Interferon 

@ ® Retinoic Acid Treated Interferon 



\ 



N 



\ 



\ 



\ 



i 



V 



-*- 



r 

o 



5 10 15 20 25 

Time (Hrs.) at 37°C 



29 



Vit amin A and Interferon Production 

Effect of Retinoic Acid on Interferon Synthesis and Cellul ar RNA 

Synthesis . 

Monolayer cultures of L-929 cells were infected with NDV as described. 
Following infection, cultures were treated with 20 ug/ml of retinoic 
acid in MEM with 3% CS and 1% DMSO. Controls received an equivalent 
amount of DMSO (1%) in culture madia. Retinoic acid or the control 
fluid either remained on the cells for 24 hours or was washed out after 
3 or 6 hours and replenished with fresh medium lacking retinoic acid 
and DMSO. Table 2 shows that retinoic acid suppressed interferon, 
production. The same amount of suppression of interferon yield resulted 
regardless of whether the retinoic acid remained on the cultures for 
the entire production period or was washed out after 3 or 6 hours. 
Moreover, when replicate noninfected cultures were treated in a similar 
fashion with retinoic acid and labeled with 10 ug/ml of -'H-uridine 
for 1 hour at 23 hours after infection, there was a 74% suppression of 
■%-uridine incorporation by cell cultures treated for 24 hours with 
retinoic acid but no suppression by cultures treated for 3 or 6 hours. 
These results indicate the suppression of interferon production was not 
correlated with the suppression of -%-uridine incorporation. Part of the 
suppression of interferon yield could be explained by inactivation of 
interferon by retinoic acid after the interferon was produced as 
previously shown (Figure 3). However, 20 u g/ml of retinoic acid in 
3% CS would inactivate only about 50% of the interferon and therefore 
would not totally explain the suppression (see Figure 4). More 



30 



TABLE 2 
Effect of Retinoic Acid on Interferon 
and Cellular RNA Synthesis 



Hour of Treatment with 
Retinoic Acid (20 ug/ml) 
Following NDV Adsorption 3 


Percent Suppression 


Interferon Yield° 
(24 hours) 


^H-Uridine 
Incorporation 


3 

6 

24 


94 
94 
98 






74 



Retinoic. acid was allowed to remain on the cells for the indicated 
times after NDV adsorption, cultures were washed to remove the 
vitamin and replenished with fresh medium and allowed to incubate 
for a total of 24 hours. 

Interferon yield was determined by pooling the supernates from 
triplicate cultures. Four dilutions of each pooled supernate 
were assayed in quadruplicate. 

H-uridine incorporation was measured after 1 hour of labeling 
at 23 hours after initial exposure to retinoic acid (non-infected 
cultures). Incorporation of ^H-uridine was determined by two 
counts of duplicate aliquots of hot 5% TCA extracts from triplicate 
cultures. 



31 



importantly, in those experiments where the retinoic acid was removed 
after 3 or 6 hours and the interferon harvested after 24 hours, one 
would expect no inhibition of interferon activity due to the retinoic 
acid since extracellular interferon did not appear until 8 hours after 
infection (vide infra). 

Effect of Retinoic Acid on L-929 Cells . 

Although the suppression of interferon production was apparently 
not correlated with the suppression of J H-uridine incorporation by 
retinoic acid, we were concerned that the suppression of interferon 
production might result from some non-specific toxicity of vitamin A. 
Studies were initiated to determine conditions for vitamin A treatment 
of cells which were relatively non-toxic. We had previously shown that 
the inhibition of interferon activity by vitamin A was dependent on the 
concentration of calf serum (Figure 2 and 4), apparently because a 
serum component bound the vitamin A. The effect of calf serum on 
suppression of 3 H-uridine incorporation, therefore, was also investigated. 

To determine the effect of CS on suppression of RNA synthesis by 
vitamin A when cells were in contact with vitamin A for 23 hours, 
confluent cell monolayers were treated at 37°C with 20 yg/ml of 
retinoic acid in MEM with 2.5, 5 or 10% CS. After retinoic acid 
treatment for 23 hours, cell cultures were washed and labeled for 1 
hour with 2 ml of MEM containing 10 yg/ml 3 H-uridine. Table 3 
shows that increasing CS concentrations prevented the inhibitory effect 
of retinoic acid on %-uridine incorporation. 



32 






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33 



Since 20 ug/ml of retinoic acid did not significantly suppress 
3 H-uridine incorporation in medium containing 10% CS, we studied the 
effects of 24 hours treatment with 5, 10, or 20 ug/ml of retinoic 
acid in MEM with 10% CS on other parameters of cell viability. As can 
be seen in Table 4, the concentrations of retinoic acid used did not 
alter: a) cell number; b) cell viability as determined by trypan 
blue dye exclusion; or c) the capacity of these cells to allow 
vesicular stomatitis virus plaque formation. In other experiments 
we have shown that L-929 cells are able to proliferate when seeded 
and grown in 20 ug/ml of retinoic acid, although they do not reach 
as high a cell density as controls (Table 5) . 

These criteria indicated that retinoic acid at the concentrations 
employed was not toxic to cells when medium containing 10% CS was 
employed. Therefore all subsequent experiments were performed with 
10% CS. 

Effect of Retinoic Acid Concentration on Interferon Production by NDV . 

Confluent cell monolayers were infected with NDV, washed and fed 
medium with 1% DMS0 or medium containing 5, 10 or 20 ug/ml of retinoic 
acid and 1% DMSO. Triplicate cultures were used for each concentration 
of retinoic acid. Supernates were harvested 24 hours after NDV in- 
fection, virus inactivated, and assayed for interferon. Table 6 shows 
that increasing concentrations of retinoic acid resulted in decreasing 
yields of interferon. Twenty ug/ml of retinoic acid was used in 
all subsequent experiments since it gave the most marked suppression 
and yet was apparently non-toxic for the cells. 



34 



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TABLE 6 
Effect of Retinoic Acid Concentration on Interferon Production 

by NDV 



36 



Retinoic Acid 

(v«g/ml) 


Interferon Yield 


PDD 50 Units/ml 


% Inhibition 


20 

10 

5 




7,500 
10,000 
15,000 
36,000 


79 
72 
58 



37 



Effect of Retinoic Acid on t he Intracellular Level of Interferon . 

Since we routinely assayed interferon in the supernates of NDV 
infected cultures, it was possible that, the suppressive action of 
retinoic acid on interferon production was due to interference with 
the release process. To test this possibility, extracellular and intra- 
cellular interferon levels were determined in retinoic acid treated and 
control cultures 24 hours after NDV infection. Treatment with retinoic 
acid reduced the amount of interferon recovered from extracts of NDV 
infected cells (Table 7). However, the ratio of extracellular/intracellular 
interferon was 6 in both the control and retinoic acid treated group. 
This data shows that retinoic acid did not suppress interferon produc- 
tion by impeding its release. 

Effect of Retinoic Acid on NDV, SFV and Poly I:C Induction of Interferon . 

The results in Table 2 indicated that retinoic acid probably sup- 
pressed an early event in the production of interferon since there 
was no difference in the degree of suppression of interferon yield if 
the retinoic acid remained in contact with the cells for the 24 hours 
of production or was removed after three hours. The earliest event 

in the production cycle of interferon is thought to be the generation 

35 

of the inducer of interferon, double stranded RNA. If retinoic 

acid interfered with virus replication, the generation of the interferon 
inducer would probably be adversely affected. Since NDV abortively 
infects L-cells we could not detect an effect of retinoic acid on NDV 
replication. SFV, however, productively infects L-cells and produces 
moderate amounts of interferon. We, therefore, investigated the effect 
of retinoic acid on SFV replication and interferon production. 



38 



TABLE 7 
Effect of Retinoic Acid on the Intracellular Level 

of Interferon 



Treatment 21 


Interferon Yield (PDD5o/ml) b 


Control 
Retinoic Acid 


Extracellular 


Intracellular 


Total 


20,800 
6,000 


3,500 
1,000 


24,300 
7,000 



a Retinoic acid (20 yg/ml) or DMSO (1%) in MEM with 10% CS was added 
to NDV infected cultures at the end of virus adsorption. 

k Culture fluids were collected 24 hours after addition of NDV. 

Triplicate supernates were pooled and were centrifuged for 15 minutes 
at 1,000 rpm to remove cells which had detached from the glass 
surface. After dialysis of the clarified supernatant fluids, they 
were assayed for the level of extracellular interferon. For the 
determination of the intracellular interferon level, the cell 
pellets from the supernates were resuspended in fresh medium and 
added back to the cells which were still adherent to the glass 
surface. The cells were frozen at -20°C, thawed and homogenized 
by 10 strokes with a Dounce homogenizer. The cell homogenates were 
dialyzed at pH 2 for 5 days and centrifuged (1,000 rpm for 15 minutes) 
prior to interferon assay. Four dilutions of each interferon sample 
were assayed in quadruplicate. 



39 



L-cell cultures were infected with NDV or SFV, as previously 
described. Cultures were refed with fresh medium containing 20 ug/ml 
of retinoic acid or 1% DMSO (controls). After three hours, cultures 
were washed to remove the vitamin and DMSO and were refed with fresh 
medium. Supernates were harvested from these cultures 24 hours after 
virus adsorption, virus inactivated by dialysis at pH 2, and assayed 
for interferon. Additionally, SFV infected cultures were assayed for 
the yield of virus. 

Table 8 shows that retinoic acid suppressed SFV induced inter- 
feron production without affecting the yield of virus. The degree of 
suppression of interferon yield was similar with both SFV and NDV. 
This data shows that retinoic acid did not suppress interferon production 
by interfering with virus replication and suggested that generation 
of the interferon inducer was not inhibited. 

In a similar experiment, L-cell cultures were induced to make 
interferon by treatment for 2 hours at 37 °C with poly I:C (10 yg/ml) 
in medium containing 100 yg/ml of DEAE dextran. After poly I:C 
treatment, cells were washed and refed with medium containing retinoic 
acid (20 yg/ml or DMSO (1%). Twenty-four hours later, supernatant 
fluids were assayed for interferon. Table 8 shows that retinoic acid 
suppressed poly I:C induced interferon production. Since poly I:C 
is a synthetic double stranded RNA molecule, the interferon inducer 
had been formed prior to interaction with the cell. In this instance, 
retinoic acid could not have interfered with generation of the inducer. 



40 



CO 

p-1 



O 

id 

ItH 

a) 

C 



IH 

o 



o 



u 

3 
-a 
C 



M 

t — i 
O 

C 
tfl 

> 
fa 
C/2 



> 
P 

C 

o 

-J 

•H 

< 



c 
C 

■H 
4J 

CD 

P4 

o 



u 
m 

IJH 

w 



Virus Yield 
PFU (xlO 8 ) 


m o 

o o 
+ i+i i 

•K 1 

o m i 

rH rH 


% Inhibition of 
Interferon Yield 


<T rH VO 

». r^ 00 


Interferon Yield 
(PDD 50 ml) 


2,600 
10,000 

260 

900 

100 
700 


Retinoic Acid 
(20 jig/ml) 


+ i +i +i 


Interferon 
Inducer 


NDV 
SFV 
Poly I:C 



tu 

O 

3 

-d 

o 
u 

a 

en 

■H 

CO 

■J 
U 



c 
c 

<u 

G 
(D 

u 



-a 
r. 



a 

fN 

Oi 

I 



U 

c 
m 
C 



tu 

> 

-H 
■M 
r-l 

C 

rj 

> 
O 
25 



41 



Kine tic s of Suppression of Interferon Yield by Retinoic Acid . 

To investigate further the period during which interferon production 
was sensitive to suppression, retinoic acid and DMSO were removed at 
various times after NDV adsorption, the cell cultures were washed and 
replenished with fresh medium without retinoic acid or DMSO. Table 9 
shows that maximum suppression of interferon production required two 
hours treatment with retinoic acid after NDV adsorption. Again there 
was no difference in the amount of suppression whether the vitamin A 
was removed after 2 hours treatment or remained on cells for 24 hours. 

Similar experiments (Figures 7 and 8) showed that even if addition 
of retinoic acid was delayed for up to 2 hours after the end of NDV 
adsorption, or poly I:C addition, there was still maximum suppression 
of interferon production. By 4 hours after NDV adsorption, or poly I:C 
addition, cells were becoming less sensitive to the suppressive effects 
of retinoic acid, and they were resistant to suppression by 8 hours. 
These data indicate that retinoic acid affected a step in the production 
of interferon which maximally occurred about 2 hou^s after NDV adsorp- 
tion or poly I:C addition. 
Kinetics of Interferon Production by Retinoic Acid Treated Cells . 

When the kinetics of interferon production in MEM with 10% CS 
were examined (Figure 9), interferon was first detected in control 
cultures 8 hours after NDV infection, while interferon was not detected 
from cultures treated with 20 ug/ml retinoic acid until 4 hours 
later. The apparent rate of synthesis of interferon was reduced 
in retinoic acid treated cultures, as was the total yield. This 
difference in apparent rates of synthesis did not result from inter- 



TABLE 9 

Effect of Time of Treatment with Retinoic Acid 
(20 yg/ml) after NDV Adsorption 



42 



Length of treatment with 
Retinoic Acid (20 yg/ml) 


Interferon Yield 




PDD units/ml 


% Inhibition 





0-0.5 hrs 
0-1 hrs 
0-2 hrs 
0-4 hrs 
0-24 hrs 


22,900 

21,000 

15,900 

6,000 

5,600 

5,600 


9 
31 
74 
76 
76 





a time is immediately following adsorption of NDV for 1 hour. 

Interferon yield was determined by pooling the supernates from 
triplicate cultures. Four dilutions of each interferon sample 
were assayed in quadruplicate. 



Figure 7. Effect of time of addition of retinoic acid after NDV 
adsorption. Monolayer cultures were infected with NDV 
as described in Materials and Methods. At the time 
intervals indicates, 20 yg/ml of retinoic acid in MEM 
with 10% CS was added. Retinoic acid remained on cells 
throughout the interferon production period. Controls 
were treated similarly with an equivalent amount of DMSO (1%) , 
Interferon yield was determined by pooling supernates 
from triplicate cultures. Four dilutions of each 
interferon sample were assayed in quadruplicate. 



44 



o 
_i 

UJ 

>- 



O 

LU 

cr 

LU 



U. 

o 

z 
o 

H 
GQ 

X 




2 4 

HOURS AFTER NDV ADSORPTION 






Figure 8. Effect of time of addition of retinoic acid after 
addition of poly I:C. Monolayer cultures were 
treated \7ith poly I:C as described in Materials and 
Methods. At the time intervals indicated 20 ug/ml 
of retinoic acid in MEM with 10% CS was added. 
Retinoic acid remained on cells throughout the 
interferon production period. Controls were treated 
similarly with and equivalent amount of DMSO (1%) . 
Interferon yields were determined by pooling supernates 
from triplicate cultures. Four dilutions of each 
interferon sample were assayed in triplicate. Aand^ 
show the results of two experiments. 



46 



1001 



Lil 



z 
o 

IxJ 

rr 

UJ 



80 



60- 



o 40- 



a\ 



o 



■z. 












o 






A 






h- 












CQ 
X 


20- 










Z 












^5 


^\ 















i 


i 


i 


i 




( 


) 2 


4 


6 


8 



HOURS AFTER ADDITION OF POLY I = C 






Figure 9. Kinetics of interferon production by control and retinoic 
acid treated cells. Cultures were infected with NDV 
as described in Materials and Methods. Cultures were 
then treated for 2 hours with retinoic acid (20 yg/ml) 
in MEM with 10% CS. Controls were treated with DMSO (1%) . 
At the times indicated triplicate cultures from both 
groups were frozen at -20°C. At the end of the experi- 
ment samples were thawed, pooled, dialyzed and assayed 
for interferon. Four dilutions of each interferon 
sample were assayed in quadruplicate. 



48 



3 5-i 



30- 



ro 

i 

o 

X 

E 
c> 
c; 



?, 



O 
5 

a. 



a 




A 9 



ference with the process of release of interferon, since we found the 
the same ratio of extracellular/intracellular interferon in both 
control and retinoic acid treated cells (Table 7). 

E ffect of Cycloheximide on Suppression of Interferon Production By 

Retinoic Acid . 

The suppression of interferon production by retinoic acid could 
be due either to a direct effect of the vitamin on an interferon control 
mechanism or indirectly through induction of another molecule. Vilcek 
et al . ^ described a paradoxical effect of inhibitors of RNA and protein 
synthesis on the production of interferon in cultures of rabbit kidney 
cells stimulated with poly I:C. In the continuous presence of cyclo- 
heximide these cells produced 3 to 10 times more interferon than control 
cultures. On the basis of kinetic studies it was concluded this effect 
was most likely explained by preferential inhibition (by cycloheximide) 
of a cellular regulatory protein (repressor) which controls interferon 

•3 7 

production. To determine if vitamin A induces the synthesis of a 
regulatory protein, we studied the effect of cycloheximide on suppression 
of interferon production by retinoic acid. 

Preliminary experiments were performed to determine a concentration 
of cycloheximide which would not inhibit interferon production. Cells 
were infected with NDV, as previously described. After adsorption of 
virus for 1 hour at room temperature 1, 5 or 10 ug/ml of cycloheximide 
in medium was added to L-cell cultures. Controls received medium 
without cycloheximide. Twenty-four hours later supernatant fluids were 
collected, dialysed and assayed for interferon. Table 10 shows that 10 and 
5 ug/ml of cycloheximide inhibited interferon production while 1 ug/ml did not, 



TABLE 10 
Effect of Cycloheximide on Interferon Production 



50 



Cycloheximide 
(ug/ml) 


% Inhibition 
of Interferon Yield 


1 

5 

10 



72 
99 



5 1. 



Therefore, 1 yg/ml of cycloheximide was employed in the following 
experiment. Cell cultures were infected with NDV. After adsorption 
of the virus, cultures were washed and refed with 20 yg/ml of 
retinoic acid in medium with or without cycloheximide. Controls 
received 1% DMSO in medium with or without cycloheximide. Twenty- 
four hours later, supernatant fluids were collected, dialysed and 
assayed for interferon. Table 11 shows that the suppressive effect 
of retinoic acid on interferon production was completely reversed by 
cycloheximide. This data suggests that retinoic acid acts indirectly 
through production of a protein which inhibits interferon production. 

Kinetics of Interferon Action in the Presence of Retinoic Acid . 

The above data indicates that retinoic acid induces a regulatory 
protein. One wonders if this protein represses all inducible cell 
functions. Interferon is thought to inhibit virus replication by 
inducing the formation of a new protein, the antiviral protein. 30,31,38 
If the retinoic acid induced protein non-specifically represses all 
inducible cell functions, one would expect it, under conditions 
of suppression of interferon production, to inhibit induction of the 
antiviral protein in the presence, of retinoic acid. 

Monolayer cultures were treated for varying periods of time with 
4 or 16 PDD50 units/ml of mouse interferon in MEM with 10% CS containing 
either 20 Ug/ml of retinoic acid or 1% DMSO. Controls received 
retinoic acid or DMSO in medium. After interferon treatment, cultures 
were washed 3 times and immediately infected with about 150 PFU of VSV. 
Forty-eight hours later cultures were stained, virus plaques enumerated 



52 



TABLE 11 

Effect of Cycloheximide on Suppression of 
Interferon Production by Retinoic Acid 



Treatment 



Retinoic Acid (20 yg/ml) 

Retinoic Acid (20 yg/ml) 
& cycloheximide (1 yg/ml) 

DMSO (1%) & cycloheximide 
(1 Pg/ml) 

DMSO (1%) 



Interferon Yield 
PDD units/ml 



2,800 
11,200 

11,200 

11,200 



% Inhibition of 
Interferon Yield 



75 




53 



and the percentage inhibition of the control plaque number determined, 
Retinoic acid caused no difference in the rate of development of 
interferon induced viral resistance when compared to controls 
(Figure 10). This data indicates that the proposed repressor protein 
induced by retinoic acid did not inhibit interferon induction 
of the antiviral protein. 



Figure 10. Kinetics of interferon action in the presence of retinoic 
acid. Monolayer cultures were treated for the time 
intervals indicated with either 4 or 16 PDD50 units/ml 
of interferon in MEM with 10% CS containing either 
20 yg/ml of retinoic acid of 1% DMSO. At the indicated 
times triplicate cultures were washed 3 times and 
infected with about 150 PFU of VSV as described 
in Materials and Methods. 



5 r > 



lOOl 



o 

it. 
o 



o 
< 



> 

CO 

> 
o 
o 



CO 
X 

2- 




PDDgQUNITS/ml of INTERFERON 



RETINOIC ACIO (20wg/ml) A 



DMSO(l%) o 



IS 
A 



1 —I 1 1 

2 4 6 8 10 24 

TREATMENT WITH INTERFERON & RETINOIC ACID OR D.MSO(HRS) 



56 



Structural Requir eme nts of Vitamin A For Suppressio n 
of Interferon Production and Inhibition of Its Action 

Different forms of vitamin A and related compounds dissolved in 
DMSO were tested to determine their effect on the antiviral activity 
of interferon. Retinoic acid, retinol, retinal, retinyl acetate, 
vitamin K-^, g-carotene and citronellol were each made 6.7 x 10~ M 
in an interferon preparation diluted 1 to 10 in MEM. Controls received 
an equivalent amount of DMSO (1%). After 24 hours at 37°C, the 
mixtures were further diluted 1 to 100 in MEM with 5% CS and assayed 
for residual interferon activity. Table 12 shows that retinoic 
acid and retinol were similarly effective at inhibiting interferon 
activity while retinal and retinyl acetate were considerably less 
inhibitory. This data suggested that the terminal group on the vitamin 
A molecule was important for the inhibitory effect on interferon 
activity. This concept is supported by the low level of inhibitory 
activity of g-carotene on interferon action, g-carotene is a dimer 
of vitamin A with a ring on both ends of the molecule. Since 
g-carotene was not very inhibitory for interferon action, it seems 
that the ring portion of the vitamin A molecule was not of much 
importance in the inhibition of interferon action. Furthermore, 
citronellol, an analogue of the side chain of retinol, was not inhibitory, 
suggesting that the conjugated double bond system was also important 
in the interaction of vitamin A with the interferon molecule. Another 
fat soluble vitamin, vitamin K,, did not inhibit interferon activity. 



57 



TABLE 12 

Structural Requirements of Vitamin A for Suppression 
of Interferon Production and Inhibition of Its Action 



COMPOUND TESTED 



% SUPPRESSION 
OF INTERFERON 



ANTIVIRAL 
ACTIVITY 



PRODUCTION 



COOH 



RETINOIC ACID 




CH 3 CH 3 CH, II , 

CH 3 ' " X 

RETINYL ACETATE 



CH3 CH3 CH^ CH3 




63 



70 



20 



24 



68 



47 



68 



40 



19 



57 



^CH 2 0H 
CITRONELLOL 



DMSO 
(CONTROL) 



58 



The same compounds were tested for their effect on interferon 
production. NDV was adsorbed to L-929 cells for 1 hour at room 
temperature. Cell cultures were then washed and received either 
retinoic acid, retinol, retinal, retinyl acetate, vitamin K^, 
3-carotene or citronellol at 6.7 x 10~ 5 M in MEM with 10% CS. 
Ten percent CS was employed because vitamin A does not inhibit 
interferon activity under these conditions. In contrast to the 
marked dependence on the form of vitamin A required for inhibition 
of interferon action, all forms of vitamin A tested suppressed 
interferon production (Table 12). This observation suggested that 
the ring portion of the vitamin A molecule was of primary importance 
for the suppression of interferon production. That 3-carotene, 
a dimer of vitamin A with a ring group at each end of the molecule, 
was suppressive for interferon production is consistent with this 
idea. Also, the lack of a suppression by citronellol, an analogue 
of the side chain of retinol, supports this concept. Vitamin K-, 
was also not very suppressive. 



DISCUSSION 

•it 
As we previously reported for human interferon, retinoic acid 

39 
was shown to inhibit the action of mouse interferon. The inhi- 
bition of antiviral activity, which was observed when retinoic acid was 
mixed with interferon and immediately assayed for interferon activity, 
apparently resulted from an interaction of retinoic acid with the 
interferon molecule. This conclusion is supported by the observation 
that treatment of interferon with retinoic acid and incubation in the 
absence of cells resulted in a loss of interferon activity when subse- 
quently measured after sufficient dilution to reduce the concentration 
of retinoic acid to a level which had no effect in the assay. In 
addition, the inhibitory effect of retinoic acid on interferon activity 
was prevented by calf serum or bovine serum albumin. 

The loss of interferon activity following addition of retinoic 
acid was characterized by a dependence on time and temperature. Tem- 
peratures above 25°C were required for a pronounced inhibitory effect. 

Recent reports have shown that human interferon has hydrophobic 
binding sites. ' »* These reports have indicated that there is no 
hydrophobic interaction between mouse interferon with w-carboxypentyl 
agarose and only slight retention of mouse interferon on albumin 
immobilized on agarose. ^ Our data is consistent with the concept 
of a hydrophobic interaction between retinoic acid and interferon. 
Davey e_t al. 41 reported that the binding of human interferon to hydro- 
phobic hydrocarbon arms covalently linked to Sepharose was critically 
dependent on the h ydrophilic head group of the hydrocarbon. We have 
also observed a critical dependence on the character of the terminal 



59 



60 



group of vitamin A for the inhibitory effect on interferon. Forms of 
vitamin A with carboxy or hydroxy groups were effective at inhibiting 
interferon while vitamin A forms with carbonyl or acetate ester groups 
were essentially ineffective. Considering the high affinity hydro- 
phobic binding sites on bovine serum albumin, ^3 ^q ca if serum depen- 
dent prevention of the inhibitory effect of retinoic acid on inter- 
feron probably results from competitive binding of the retinoic acid 
to albumin and other serum proteins. Bovine serum albumin can be sub- 
stituted for calf serum in preventing the effect of retinoic acid on 
interferon. 

We have assumed that serum or bovine serum albumin interacts with 
vitamin A to prevent its binding to interferon. It is possible, of 
course, that serum interacts with interferon to prevent its binding 
to vitamin A. However, considering the high affinity that albumin 
has for fatty acids ^ 3 and the low affinity for mouse interferon, ^ 2 
it is more likely that serum interacts with vitamin A to prevent 
vitamin A from binding to interferon. 

If retinoic acid exerts its inhibitory effect by binding to hydro- 
phobic regions on interferon molecules, it would seem this binding must 
be of relatively high affinity as compared with calf serum since the 
molar concentration of protein in serum should be considerably higher 
than the molar concentration of interferon. Since mouse interferon 
seems to have hydrophobic regions, this data suggested that mouse 
interferon might be purified by hydrophobic chromatography. Unfortunately, 
there are no means by which to couple the ring portion of the vitamin 
A molecule to a solid matrix. Coupling by the ring portion would seem 



61 



essential since the side chain and hydrophilic end of the vitamin A 
molecule appears to be most important in the interaction with the 
interferon molecule. In fact, interferon did not bind to retinoic 
acid coupled by its carboxy group to Sepharose. Recently, however, 

mouse interferon has been greatly purified by hydrophobic 

44 
chromatography. 

We have also shown a suppressive effect of vitamin A on interferon 
production. Our in vitro suppression is in agreement with a report 
of an in vivo suppression of interferon production by vitamin A. 
Based on our previous findings the first possible explanation was that 
the suppressive effect might result from inactivation of interferon 
by vitamin A after it was produced. This was eliminated by several 
lines of evidence: removal of retinoic acid after 2 hours did not cause 
any less suppression than its continued presence throughout the interferon 
production period (24 hours) ; interferon production was suppressed 
in a calf serum concentration (10%) in which 20 pg/ml of retinoic 
acid does not inactivate interferon activity; and a form of vitamin A 
(retinal) which was very effective at suppressing interferon production 
was ineffective at inactivating interferon activity. The enhanced 
suppression of interferon production observed in 3% CS, however, might 
be accounted for by inactivation of interferon by retinoic acid. 

A second possible explanation was that the suppressed production 
resulted from non-specific toxicity. This possibility was eliminated 
since in 10% CS the concentration of vitamin A employed was non-toxic 
as measured by: cell number; cell viability as determined by trypan 
blue dye exclusion; plaque forming ability of VSV; H-uridine incorpora- 
tion; and the ability of cells to proliferate in vitamin A. 



62 



We have found that retinoic acid treatment reduced the amount 
of interferon recovered from lysed NDV infected cells and that the 
ratio of extracellular/intracellular interferon is apparently 6 in 
both control and retinoic acid treated cultures 24 hours after NDV 
infection. This data indicates that retinoic acid does not impede 
the release of interferon from the synthesizing cells. 

Unimpaired protein synthesis is, of course, a prerequisite to 
maximal interferon production. Known inhibitors of protein synthesis, 
such as puromycin and p-fluorophenylalanine, are suppressive 
throughout the period of interferon production. If retinoic acid 
acted by inhibition of protein synthesis it should also be active 
throughout the cycle of interferon production. Contrary to this, we 
have shown that addition of retinoic acid 8 hours after interferon 
induction, the time at which interferon is first detectable, did not 
suppress the final yield of interferon. Furthermore, it seems unlikely 
that vitamin A acted by inhibition of protein synthesis since cells 
can proliferate in 20 Ug/ml of retinoic acid and VSV and SFV 
can grow normally in vitamin A treated cells. 

This work also demonstrates that vitamin A suppressed an early 
event in the production of interferon. The earliest event in the 

production cycle of interferon is thought to be the generation of the 

35 
inducer, double stranded RNA. ' However, retinoic acid inhibited 

interferon production in response to SFV without inhibiting the yield 

of virus. Since double stranded RNA is a by product of virus repli- 

48 
catxon and virus replication was not inhibited, it seems improbable 

that retinoic acid interfered with the generation of the inducer. 



63 



Furthermore, retinoic acid suppressed poly I:C induced interferon 
production. Since poly I:C is a synthetic double stranded RNA molecule, 
the. interferon inducer had been formed prior to interaction with the 
cell and would, therefore, preclude interference with generation 
of the inducer. 

Specific m-RNA synthesis must also be an early event in the production 
of interferon. There are several reports concerning the effects of 
adding actinomycin D at different times after induction of interferon 

synthesis and these have shown that interferon messenger RNA synthesis 

j • i_- r „ -,-■ • - ■ * ■ 49,50,51 

xs completed within a few hours following virus infection. 

Although total RNA synthesis was not altered in our system, the kinetics 

of the suppressive effect of vitamin A on interferon production are 

consistent with the suppression of interferon messenger RNA synthesis 

since the maximum effect occurred about 2 hours after adsorption of 

NDV or addition of poly I:C and the kinetics of the effect are very 

49 
similar to those seen with actinomycin D. If only a few messenger 

RNA species were suppressed, one would not detect a difference in total 
RNA. This would be an important difference between the two systems 
(actinomycin D vs. vitamin A), since actinomycin D caused a marked 
inhibition of total RNA synthesis while vitamin A did not. 

Vilcek and Ng have postulated that interferon synthesis in 
rabbit kidney cells is controlled by a cellular repressor. Their con- 
clusion is based on the increased yield of interferon in the continued 
presence of low concentrations of cycloheximide. This data indicated 
a preferential inhibition of the repressor of interferon production by 
cycloheximide. If rabbit cultures induced to synthesize interferon 



u 



and exposed to cycloheximide for 4 hours were then treated with actino- 
mycin D and the cycloheximide hlock reversed, an even larger increase 
in interferon yield resulted, termed superinduction. It was virtually 
impossible that further synthesis of interferon m-RNA could have occurred 
after treatment with actinomycin D (5 ug/ml). Actinomycin D, there- 
fore, inhibited a cellular function which otherwise would have pre- 
maturely terminated the translation of interferon m-RNA. Hence, the 
repressor aDparently acted at the post-transcriptional level. We 
have observed that interferon production by L-cells is resistant to 
1 ug/ml of cycloheximide. This finding is not surprising since 
interferon is induced by double stranded RNA which itself inhibits 
protein synthesis. -" When cell cultures were treated with this 
concentration of cycloheximide and with retinoic acid (20 ug/ml) 
there was no suppression of interferon production by the vitamin, 
whereas vitamin A treatment alone caused a 75% inhibition of inter- 
feron yield. Although other interpretations are possible, one explanation 
of the data is that retinoic acid indirectly suppressed interferon 
production through induction of a new protein. The synthesis of 
this protein must, therefore, be more sensitive to cycloheximide than 
is interferon synthesis. If retinoic acid had only increased the speed 
or amount of production of a protein already present or normally 
induced, one would have expected cycloheximide alone to have increased 
the yield of interferon as compared to the control. Whether this 
postulated repressor acts at the transcriptional or post-transcriptional 
level is not presently known. However, the kinetics of the suppressive 
effect of retinoic acid suggests that transcription of the m-RNA for 



65 



interferon is suppressed. This idea is based on the finding that addition 
of retinoic acid at 8 hours after interferon induction did not suppress 
interferon production. Even considering a possible 2 hour requirement 
for the maximum suppressive effect of retinoic acid, this would be only 
10 hours into the interferon production cycle. Ten hours post induction 
is well before the bulk of the interferon has been synthesized. Since 
interferon release into the culture fluid very closely follows trans- 
lation of the interferon messenger RNA, this data indicates that 
translation is not suppressed. 

Interferon acts through the induction of a new protein, the anti- 
viral protein. 30,31,38 The postu i atec j vitamin A induced repressor 
is probably somewhat specific in its action since retinoic acid did 
not interfere with the generation of the antiviral state in cells treated 
with interferon. Furthermore, vitamin A did not suppress SFV replication 
which also requires new protein synthesis. It is tempting to speculate 
the vitamin A may induce a regulatory protein which specifically suppresses 
induction of certain messenger RNAs, one of which is the interferon 
messenger RNA. 

Numerous studies of the action of vitamin A have yielded a wealth 
of information. For example, vitamin A can: Stimulate growth of 
chick heart fibroblasts; 55 alter differentiation of epithelial 
cells; ' suppress induction and growth of tumors in response to 
chemical carcinogens; °' 7 and alter glycoprotein synthesis. 27 > 28 
Although all of these studies indicate a role for vitamin A in gene 
expression, none of them have shown a specific site of action. An 



66 



intracellular retinoic acid binding protein has recently been dis- 

, 56,57 
covered and has led to speculation that retinoic acid may 

bind to this protein and act directly at the gene level, similar to 
a steroid-steroid receptor complex. Our data suggests that the site 
of action of vitamin A is at the gene level since it appears that 
interferon production is suppressed at the transcriptional level by 
a vitamin A induced protein. Interestingly, superinduction of inter- 
feron has only been reported in primary (normal) cell cultures. 52 > 58 ' 59 
Superinduction by cycloheximide and actinomycin D might, therefore, 
be thought of as resulting from a block in a control mechanism for 
interferon which is operational in normal cells. We have not observed 
superinduction in L-cells (transformed cells) which implies they 
either do not have or do not express the normal regulatory mechanism for 
interferon production. If this is the case, L-cells, once induced, 
produce a maximal amount of interferon whereas normal cells do not. 
The suppression of interferon production by vitamin A might then be 
interpreted as a restoration of regulation of interferon production 
with the 10 to 25% of interferon which is not suppressed representing 
the amount produced by normal cells. The vitamin A treated cell 
would, in a sense, be normal and the cycloheximide block of the 
vitamin A induced suppression of interferon production could be 
interpreted as superinduction. In other words, vitamin A might 
restore control of the production of a regulatory protein for 
interferon production. Supportive of this hypothesis is the observation 
that L-cells, since they are transformed, have lost control of 
proliferation and vitamin A can apparently restore control of their 



67 



proliferation. 9 Hence, we wish to propose that vitamin A controls, 
or is necessary for regulation of, inducible gene expression and 
that control of the production of interferon is one manifestation of 
this potential. The system might, therefore, provide a model for the 
study of the action of vitamin A at the molecular level. 

Finally, these results have illustrated that vitamin A can 
suppress both interferon action and production. The inhibitory 
effects, however, appear to result from different mechanisms. The 
inhibition of interferon activity by vitamin A seems to be due to an 
effect of vitamin A on the interferon molecule, while the suppressive 
effect on interferon production is clearly due to an effect of vitamin 
A on the cell which has been induced to make interferon. It is 
interesting that the different mechanisms of action of vitamin A on 
interferon action and production apparently occur by different 
moieties of the vitamin A molecule „ 60 Inhibition of interferon 
action seems to be most dependent on the side chain of the vitamin 
A molecule. The side chain apparently requires a conjugated double 
bond system with a hydrophilic terminal group (hydroxy group for retinol 
and carboxy group for retinoic acid) to inhibit interferon action. 
In contrast, the ring portion of the vitamin A molecule seems to be 
most important for the suppression of interferon production. 



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blindness. Proc. Natl. Acad. Sci. U.S.A. 44:648. 

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Am. J. Path. 38:335. 

3. Mason, K.E. 1935. Foetal death, prolonged gestation, and 

difficult parturition in the rat as a result of vitamin A- 
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4. Wolbach, S.B. and Howe, P.R. 1925. Tissue changes following 

deprivation of fat-soluble A vitamin. J. Exp. Med. ^2:753. 

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of embryonic chicken skin explanted at different stages of 
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1967. Experimental cancer of the lung. Inhibition by vitamin 
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and squamous cell tumors. Cancer 20:857. 

7. Bollag, W. 1972. Prophylaxis of chemically induced benign 

and malignant epithelial tumors by vitamin A acid (retinoic 
acid). Eur. J. Cancer _8:689. 

8. Felix, E.L., Loyd, B. and Cohen, M.H. 1975. Inhibition of 

the growth and development of a transplantable murine 
melanoma by vitamin A. Science 189:886. 

9. Dion, L.D., Blalock, J.E. and Gifford, G.E. 1976. Vitamin A- 

induced density dependent inhibition of L-cell proliferation. 
J. Natl. Can. Inst. Submitted for publication. 

10. Moore, T. 1957. "Special Topics." In: Vitamin A, Elsevier 

Publishing Co., Amsterdam, p. 459. 

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Biol. Med. Submitted for publication 



BIOGRAPHICAL SKETCH 

James Edwin Blalock was born in Madison, Florida, on September 29, 
1949. He attended the public schools in Madison County and graduated 
from Madison High School in June, 1967. From September, 1967, to June, 1969, 
he attended North Florida Junior College, Madison, Florida and received 
an Associate of Arts degree magna cum laude in June, 1969. The re- 
mainder of his undergraduate work was performed at the University of 
Florida, Gainesville, Florida where he was awarded a Bachelor of Science 
degree in December, 1971, with a major in Microbiology. From January, 1972, 
until the present time he has pursued the degree of Doctor of Philosophy 
in the Department of Immunology and Medical Microbiology, College of 
Medicine, University of Florida. During this time his research 
received First Place among Junior Investigators at the 1975 meeting of 
the Southeastern Section of the Society for Experimental Biology and 
Medicine at Duke University, Durham, North Carolina. In June, 1976, 
he received the annual Graduate Student Research Award from the University 
of Florida Chapter of the Society of the Sigma Xi. He was supported 
by NIH Training Grant 5TI AI-0128 and NIH Research Grant AI-10900 
during his graduate training. 

Following his graduation from the University of Florida in June, 1976, 
he accepted a position as a postdoctoral fellow in the laboratory of 
Dr. Samuel Baron, Chairman of the Department of Microbiology at the 
Medical Branch of the University of Texas at Galveston, Texas. 



/;; 



74 



As a result of his research while a graduate student at the University 
of Florida, J.E. Blalock published or was in the process of publishing 
at the time of graduation the following papers: 

1. Blalock, J.E. and Gifford, G.E. 1974. Effect of Aquasol A, vitamin 

A and Tween 80 on vesicular stomatitis virus plaque formation 
and on interferon action. Arch. ges. Virusforsch. 45:161-164. 

2. Blalock, J.E., Tullish, J. and Gifford, G.E. 1975. Effect of Tween 

80 and Aquasol A on virus plaque formation. Inf. and Imm. 
12:490-494. 

3. Blalock, J.E. and Gifford, G.E. 1975. Inhibition of interferon 

action by vitamin A. J. Gen. Virol. 29:315-324. 

4. Blalock, J.E. and Gifford, G.E. 1976. Suppression of interferon 

production by vitamin A. J. Gen. Virol. in press. 

5. Blalock, J.E. and Gifford, G.E. 1976. Comparison of the suppression 

of interferon production and inhibition of its action by 
vitamin A and related compounds. Proc. Soc . Exp. Biol. Med. 
submitted for publication. 

6. Dion, L.D., Blalock, J.E. and Gifford, G.E. 1976. Vitamin A 

induced density dependent inhibition of L-cell proliferation. 
J. Natl. Can. Inst. submitted for publication. 

7. Blalock, J.E. and Gifford, G.E. Effect of vitamin A and a nonionic 

surface active agent on vesicular stomatitis virus plaque for- 
mation. Abs. Ann. Meeting Amer. Soc. Microbiol., 1973, p. 205. 

8. Blalock, J.E. Effect of vitamin A and a nonionic surface active 

agent on virus replication and on interferon action. Abs. 
Southeastern Sectional Meeting Society for Experimental Biology 
and Medicine, 1973, p. 16. 

9. Blalock, J.E. Suppression of interferon production by vitamin A. 

Abs. Southeastern Sectional Meeting Society for Experimental 
Biology and Medicine, 1975. 



1 certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is fully 
adequate, in scope and quality, as a dissertation for the degree of 
Doctor of Philosophy. 



> 



George E. Gifford, Ph.D., Chairman 
Professor of Immunology and 
Medical Microbiology 





I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is fully 
adequate, in scope and quality, as a dissertation for the degree of 
Doctor of Philosophy. 




(a) . ^44 a^L (J 

ands, Jr. , M.D. V 



Shands, Jr. , M.D. 
^ofessor of Immunology and 
Medical Microbiology 



I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is fully 
adequate, in scope and quality, as a dissertation for the degree of 
Doctor of Philosophy. 



&*4A 





L. William Clem, Ph.D. 
Professor of Immunology and 
Medical Microbiology 



I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is fully 
adequate, in scope and quality, as a dissertation for the degree of 
Doctor of Philosophy. 




Donna H. Duckworth, Ph.D. 
Associate Professor of Immunology 
and Medical Microbiology 



I certify that I have read this study and that in my opinion it 
conforms to acceptahle standards of scholarly presentation and is fully 
adequate, in scope and qualify, as a dissertation for the. degree of 
Doctor of Philosophy. 



ineth Ley, Ph.D. , AatLerant 



Kenneth Ley, Ph.D. , Asfiie'Eant 
Professor of Veterinary Science 



I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is fully 
adequate, in scope and quality, as a disserta tion for the degree of 
Doctor of Philosophy. 




'Zjl Qt.tf&u^ 



Paul A. Klein, Ph.D., Associate 
Professor of Pathology 



I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is f,ully 
adequate, in scope and quality, as a dissertation for the 
Doctor of Philosophy. 




// Parker A. Skall 

Professor and Intei 

Immunology and Medical Microbiology 



This dissertation was submitted to the Dean of the College of Medicine 
and to the Graduate Council, and was accepted as partial fulfillment of 
the requirements for the degree of Doctor Philosophy. 



June, 1976 




Dean, 



lcxne 



Dean, Graduate School